ARTICLE https://doi.org/10.1038/s41467-019-09636-6 OPEN MKL1-actin pathway restricts chromatin accessibility and prevents mature pluripotency activation Xiao Hu1,2, Zongzhi Z. Liu1,2,3, Xinyue Chen1,2, Vincent P. Schulz4, Abhishek Kumar 5, Amaleah A. Hartman1,2, Jason Weinstein1,2, Jessica F. Johnston 1, Elisa C. Rodriguez1, Anna E. Eastman1,2, Jijun Cheng2,6, Liz Min1, Mei Zhong1,2, Christopher Carroll1, Patrick G. Gallagher3,4,6, Jun Lu2,6, Martin Schwartz 5, Megan C. King1, Diane S. Krause1,2,7 & Shangqin Guo1,2 1234567890():,; Actin cytoskeleton is well-known for providing structural/mechanical support, but whether and how it regulates chromatin and cell fate reprogramming is far less clear. Here, we report that MKL1, the key transcriptional co-activator of many actin cytoskeletal genes, regulates genomic accessibility and cell fate reprogramming. The MKL1-actin pathway weakens during somatic cell reprogramming by pluripotency transcription factors. Cells that reprogram efficiently display low endogenous MKL1 and inhibition of actin polymerization promotes mature pluripotency activation. Sustained MKL1 expression at a level seen in typical fibro- blasts yields excessive actin cytoskeleton, decreases nuclear volume and reduces global chromatin accessibility, stalling cells on their trajectory toward mature pluripotency. In addition, the MKL1-actin imposed block of pluripotency can be bypassed, at least partially, when the Sun2-containing linker of the nucleoskeleton and cytoskeleton (LINC) complex is inhibited. Thus, we unveil a previously unappreciated aspect of control on chromatin and cell fate reprogramming exerted by the MKL1-actin pathway. 1 Department of Cell Biology, Yale University, New Haven, CT 06520, USA. 2 Yale Stem Cell Center, Yale University, New Haven, CT 06520, USA. 3 Department of Pathology, Yale Cancer Center, Yale University, New Haven, CT 06520, USA. 4 Department of Pediatrics, Yale University, New Haven, CT 06520, USA. 5 Department of Medicine (Cardiology), Yale University, New Haven, CT 06520, USA. 6 Department of Genetics, Yale University, New Haven, CT 06520, USA. 7 Department of Laboratory Medicine, Yale University, New Haven, CT 06520, USA. Correspondence and requests for materials should be addressed to S.G. (email: [email protected]) NATURE COMMUNICATIONS | (2019) 10:1695 | https://doi.org/10.1038/s41467-019-09636-6 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09636-6 he nucleus orchestrates characteristic gene expression The expression of many actin cytoskeletal genes is controlled Tprograms often by modulating chromatin accessibility, by the transcriptional co-activator, MKL1 (Megakaryoblastic thereby determining cellular identity. Chromatin accessi- Leukemia 1, MRTF-A), in complex with the Serum Response bility is best known to be catalyzed by biochemical activities from Factor (SRF) via binding to the CArG consensus sequence various nuclear-localized epigenetic remodeling enzymes1,2. (Supplementary Fig. 1d)19,20. The transcriptional activity of Whether the nucleus and chromatin accessibility is controlled by MKL1 is primarily controlled by its cytoplasmic-nuclear shuttling elements external to the nucleus, such as those conducting the via binding to monomeric actins21. To determine whether the biomechanical cues, is largely unexplored. subcellular localization of MKL1 changes during reprogramming, The nucleus is physically connected with the cytoskeleton via we tracked the localization of a virally expressed MKL1-GFP the linker of the nucleoskeleton and cytoskeleton (LINC) com- fusion protein22 in mouse embryonic fibroblasts (MEFs) under- plex, a highly conserved nuclear envelope bridge consisting of going reprogramming, during which all cells expressed OKSM Sun proteins and Nesprins3–5. It is known that the cytoskeleton when doxycycline (Dox) was added23. While many cells displayed and the LINC system are responsible for physically positioning nuclear MKL1-GFP at the onset of reprogramming, as expected the nucleus inside the cell and for deforming it in response to for fibroblasts grown in the presence of serum24, its subcellular mechanical signals6–9. Mechanical strains on the nucleus medi- localization became predominantly cytoplasmic when reprogram- ated by the actomyosin system could be severe enough to cause ming progressed (Fig. 1a, b), indicating reduced MKL1 activity. nuclear envelope herniation or rupture7,10–12. Strains from The altered MKL1 localization was paralleled by significant polymerized actins have also been reported to cause transcrip- reduction in F-actins, as determined by phalloidin staining tional repression13. These evidences suggest that in addition to (Fig. 1c). At gene expression level, the resulting induced regulating the physical state of the nucleus, the cytoskeleton pluripotent stem cells (iPSCs) showed reduced expression of might also be able to modify the nucleus’ biochemical state. many actin cytoskeletal genes, as well as low levels of endogenous However, the extent and nature of this modulation, as well as the MKL1 as compared to MEFs (Fig. 1d). Of note, mouse embryonic underlying mechanism remain unclear. stem cells (mESCs) displayed similarly low expression and We explored these questions using somatic cell reprogramming polymerization of actins (Fig. 1c, d), suggesting that the MKL1- into pluripotency as a model system. Pluripotent stem cells dis- actin pathway activity is low in pluripotent cells. Overall, MKL1 play highly open/accessible chromatin14,15, which can be activity and actin cytoskeletal genes are decreased during somatic experimentally induced from somatic cells of much reduced cell reprogramming into pluripotency. genomic accessibility. During reprogramming, when the tran- scription factors Oct4/Sox2/Klf4 (OSK) are first expressed in fibroblasts, they fail to bind the authentic pluripotency sites even MKL1 elevates cytoskeletal genes and blocks pluripotency.To though they are considered to possess pioneer activity16,17. The test whether the reduction in actin and related genes merely mark promiscuous binding by these pioneer factors to the somatic the cells progressing toward pluripotency, or functionally control genome suggests that chromatin accessibility might be initially cell fate transition into pluripotency, we sustained the level of constrained by mechanisms that are particularly active in somatic actin cytoskeletal genes by expressing a constitutively active cells. Here, we report that the actin cytoskeleton, and the main mutant MKL1 (caMKL1)21, or the control vector, in repro- transcription factor complex controlling its abundance, MKL1/ grammable MEFs that also contain an Oct4:GFP knock-in SRF, limits cell fate reprogramming by regulating global chro- reporter25 (Fig. 2a). The choice of expressing MKL1, instead of matin accessibility. High MKL1 activity generates excessive specific actin genes, is because MKL1/SRF concertedly upregulate actins, polymerization of which leads to a significantly reduced many actin and related genes19,20, as is the case represented by nuclear volume via a mechanism involving the LINC complex. the slow cycling cells (Supplementary Fig. 1b). A constitutively Within the small nucleus, chromatin accessibility is impaired and nuclear localized MKL1 was necessary because the wild type endogenous pluripotency fails to establish. Overall, we propose (WT) protein is inhibited by G-actin21, resulting in cytoplasmic that the actin cytoskeleton is capable of constraining global localization (Fig. 1a, b) and ineffective upregulation of actin genes chromatin accessibility. The highly accessible pluripotent genome as reprogramming progresses. The MKL1 mRNA level yielded by is accommodated by a weak actin cytoskeleton. ectopic expression was comparable to that of bulk MEFs (Fig. 2b). In the presence of Dox, both caMKL1-expressing and empty vector control MEFs gave rise to alkaline phosphatase (AP)+ Results colonies of ESC/iPSC-like morphology (Fig. 2c), indicating suc- Reprogramming is accompanied by reduced actin-MKL1 cessful early reprogramming. However, unlike control iPSCs, activity. Our previous work revealed that somatic cells with an caMKL1-expressing cells failed to activate the endogenous plur- ultrafast cell cycle are efficiently reprogrammed via ectopic ipotency reporter Oct4:GFP, even after prolonged Dox treatment expression of Oct4/Sox2/Klf4/Myc (OSKM), a property that (Fig. 2d, Supplementary Fig. 1e). They remained strictly depen- allows for their prospective isolation18. The fast cycling cells were dent on exogenous reprogramming factors, as Dox withdrawal morphologically distinct as compared to their slower cycling induced their rapid reversion to a fibroblastic morphology and counterparts (Supplementary Fig. 1a). While the slow cycling cells loss of AP expression (Fig. 2e), suggesting that sustained MKL1 had a typical fibroblastic appearance, the fast cycling cells activity inhibits the activation of mature pluripotency. appeared light-reflective and minimally spread (Supplementary Examination of reprogramming from additional somatic cell Fig. 1a). This morphological distinction suggests underlying dif- types, i.e. hematopoietic cells, confirmed the inhibitory effect by ferences in the level and/or conformation of their cytoskeletal caMKL1 (Supplementary Fig. 2). Specifically, granulocyte and components. Indeed, the fast cycling cells displayed reduced macrophage progenitors (GMPs), which reprogram efficiently as expression in many actin